The entire set of cellular proteins is generated through translation of mRNAs by ribosomes. The identity and amount of the proteins that a cell synthesizes are critical parameters in determining the physiological state of the cell. Protein synthesis is frequently not proportional to mRNA levels, mainly because translation is often tightly regulated; indeed, many critical controls in gene expression occur at the level of translation (Sonnenberg et al., Cell 136(4):731-745 (2009); Selbach et al., Nature 455(7209):58-63 (2008); Baek et al., Nature 455(7209):64-71 (2008)). Under specific conditions (such as heat shock, starvation, availability of iron, etc.), translational controls ensure that synthesis of specific cellular proteins is quickly turned on or off. Translational controls are particularly prominent in systems in which transcription is inhibited, such as in early embryonic development before the onset of zygotic transcription. Furthermore, translation of many proteins is spatially localized, as underscored by the finding that the majority of mRNAs in Drosophila embryos display distinct subcellular patterns (Lecuyer et al., Cell 131(1):174-187 (2007)).
Understanding how gene expression is regulated at the level of translation, spatially and temporally, requires tools for visualizing and identifying nascent polypeptide chains. The current method used for this purpose relies on the biosynthetic incorporation of azide- or alkyne-bearing methionine (Met) analogs such as azidohomoalanine (Aha) (Dieterich et al., Proc. Natl. Acad. Sci. USA 103(25):9482-9487 (2006); Link et al., J. Am. Chem. Soc. 125(37):11164-11165 (2003)) or homopropargylglycine (Hpg) (Beatty et al., Angew. Chem. Int. Ed. Engl. 41(14):2596-2599 (2002)). The resulting azide or alkyne-labeled proteins can be detected by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (Rostovtsev et al., Angew Chem. Intl. Ed. Engl. 41(14):2596-2599 (2002); Tornoe et al., J. Org. Chem. 67(9):3057-3064 (2002); Wang et al., J. Am. Chem. Soc. 125(11):3192-3193 (2003)) with reagents for fluorescence detection (Beatty et al., Angew. Chem. Int. Ed. Engl. 41(14):2596-2599 (2002)) or for affinity purification and identification by mass spectrometry (Dieterich et al., Proc. Natl. Acad. Sci. USA 103(25):9482-9487 (2006)). Though simple and robust, this method has a number of drawbacks. Cells prefer Met over Aha or Hpg by a factor of about 500 (Beatty et al., Angew. Chem. Int. Ed. Engl. 41(14):2596-2599 (2002)), which means cultured cells need to be labeled with Aha or Hpg in Met-free media; this limitation precludes the use of Aha and Hpg to study protein synthesis in whole animals. To be incorporated into proteins, Aha and Hpg need to be activated as aminoacyl-tRNAs, a step which limits the temporal resolution of this method. Finally, this method generates full-length Aha- or Hpg-labeled proteins, not nascent polypeptide chains. Improved labeling techniques are needed for the study of nascent proteins in vivo, in particular methods that are rapid, sensitive, and work well in whole organisms.